1. Field of the Invention
This invention relates to radioimmunoassay measurement of the concentration of mammalian parathyroid hormone and certain fragments thereof in biological or other fluids.
2. Description of the Prior Art
Human parathyroid hormone (i.e. intact hPTH) is an 84 immuno acid sequence with a major role in maintaining the constancy (homeostasis) of calcium in cells and extracellular fluid, despite marked variations in calcium intake and excretion. Parathyroid peptides (both intact hPTH and certain hPTH fragments) are secreted from the parathyroid glands in response to a lowering of serum calcium; and, in the absence of parathyroid peptide secreting tumors, high levels of serum calcium inhibit secretion by the glands. Measuring parathyroid peptides in human serum has proven clinically to be an extremely helpful and efficient tool in the differential diagnosis and management of hypercalcemia; most notably, radioimmunoassay (i.e. RIA) can help to diagnose tumors and hyperplasia of the parathyroid glands. RIA detection of high blood levels of PTH peptides can establish that excess PTH secretion is causing hypercalcemia. RIA's can also be useful in localizing hyperfunctioning parathyroid tissue by assaying samples obtained via venous catheterization. PTH RIA is also useful for the diagnosis and management of hypocalcemia. With rare exceptions, hypocalcemic persons with deficient PTH (hypoparathyroidism) usually have subnormal blood concentrations of PTH peptides. Measurement of the concentration of PTH peptides in blood serum is frequently used to assess the status of renal osteodystrophy in renal failure patients on chronic dialysis. Serum PTH levels are often markedly elevated in patients with renal failure due to chronic negative calcium balance, hypocalcemia, and consequent secondary hyperparathyroidism.
In clinical parathyroid literature, a set of nomenclature has become common. N-terminal hPTH is the 1-34 amino acid sequence of hPTH. Antisera having a substantially high affinity for this sequence are referred to as N-terminal antibodies. Such antibodies will generally also have a high affinity for any sequence of amino acids incorporating the 1-34 amino acid sequence. Thus, such antibodies will have a high affinity for intact hPTH (i.e. hPTH.sup.1-84) as well as N-terminal hPTH. C-terminal hPTH is the 35-84 amino acid sequence. Antisera having a substantially high affinity for the 35-84 sequence are referred to as C-terminal antisera. It is now well established that only small amounts of intact hPTH and even less free N-terminal hPTH are present in peripheral human serum. The main circulating hPTH peptide is C-terminal hPTH. Accordingly, RIA's are generally directed at C-terminal hPTH where the purpose is diagnosing hyperthyroidism and the other maladies recited above.
Prior art RIA's which measure the concentration of C-terminal hPTH in human serum are common; however, very few are available commercially. The commercially available assays generally involve the double antibody method. In brief, C-terminal hPTH (and lesser quantities of intact hPTH) present in human serum compete with a radioactively labeled peptide (generally radioactively labeled beef PTH.sup.1-84) for a particular first antibody, thereby inhibiting the binding of the first antibody to the radioactively labeled peptide. As a result of the competitive inhibition, the ratio of first antibody bound labeled peptide to free labeled peptide diminishes as the concentration of C-terminal hPTH (and intact hPTH) increases. As a convenient method to circumvent the difficult task of separating bound labeled peptide from free labeled peptide, a second antibody is added that has a substantially high affinity for the first antibody and which forms a precipitate when bound to the first antibody bound labeled peptide. The quantity of second antibody added is generally only sufficient to bind a portion of first antibody found radioactively labeled peptide. The ratio of precipitated radioactively labeled peptide to non-precipitated radioactively labeled peptide similarly diminishes as the concentration of C-terminal hPTH (and intact hPTH) increases. The concentration of C-terminal hPTH (and intact hPTH) in an unknown sample is obtained by comparing the inhibition observed with the inhibition produced by known amounts of C-terminal hPTH (or intact hPTH), as presented in a standard curve. The concentration of C-terminal hPTH is generally represented as hPTH.sup.1-84 equivalents. That is, the concentration in nanograms/ml (ng/ml) of serum is reported as if C-terminal hPTH were intact hPTH.
Most prior art double antibody PTH RIA's face a common difficulty in measuring the concentration of C-terminal hPTH: they often give a spurious value because of nonspecific interaction (NSI) of the labeled PTH peptide with serum proteins. That is, the labeled peptide is thought to react with various proteins present in the serum sample with the result that the first antibody, which ordinarily has a high affinity for the labeled PTH peptide, has a low or no affinity for the product of the reaction of the labeled PTH peptide with serum proteins.
In some prior art double antibody PTH RIA's, the standardized reagents used to prepare the standard curve do not contain human serum (Isotex Diagnostics, Friendswood, Texas 77546). In this case, standards are generally prepared by dissolving appropriate quantities of PTH peptides, whether human, beef, or of other sources, in an appropriate non-serum buffer. The NSI for the unknown sample of serum will depress the value of the ratio of precipitated to non-precipitated labeled PTH peptide, while there will be no similar depression for the standard reagents. This is so because the latter do not contain human serum and therefore the serum proteins which cause NSI are absent. Thus, the assay will indicate a concentration that is higher than the actual concentration. The error injected into assay results by NSI can be sufficiently great, on the order of several ng's/ml of hPTH.sup.1-84 equivalents, to cause a mischaracterization of an unknown sample. That is, an assay of serum taken from a person with normal parathyroid glands could indicate hyperparathyroid disease.
Some prior art double antibody PTH RIA's seek to eliminate the effect of NSI on assay results. One prior art method (Arnaud, C. D., et al., 50 J. Clin. Invest. 21 (1971)) incorporates serum from hypoparathyroid patients or pooled serum stripped of PTH peptides in the standard reagents used to prepare the standard curve. This attempt to adjust for the effects of NSI on assay results is not wholly satisfactory. The serum added to the standard reagents is not the unknown serum to be tested. The concentration of serum peptides which give rise to NSI may vary significantly from person to person. Thus, depending upon whether the NSI for the unknown sample is relatively greater or less than the NSI for the serum incorporated in the standard reagents, the assay will indicate respectively concentrations lower or higher than the actual concentration. The error injected into this type of prior art assay can also be sufficiently great to mischaracterize an unknown sample.
Another PTH assay approach seeks to eliminate the effects of NSI on assay results by performing the RIA on significantly diluted serum. Diluting the serum in this manner does reduce NSI. However, while reducing the effect of NSI on assay results, diluting the serum generally reduces the sensitivity of the RIA to the degree that the RIA cannot detect the concentrations of C-terminal hPTH necessary to distinguish normal from abnormal functioning parathyroids.
At least one prior double antibody PTH RIA has adjusted for the effects of NSI while retaining the necessary sensitivity of the assay (PTH II, Immuno Nuclear Corp., Stillwater, Minn. 55082). In this RIA, an unknown sample of serum is divided into two portions. Intact PTH and C-terminal hPTH are removed from one of the two portions, leaving the serum proteins behind. Removing the intact hPTH and C-terminal hPTH in this manner is accomplished by binding the first antibody to a gel or resin to create a specific solid phase absorbent for intact PTH and C-terminal hPTH. However, removing the intact hPTH and C-terminal hPTH in this manner requires a full cycle of incubation of the portion of the serum to be stripped. Following this cycle of incubation, both the untouched portion of serum and the stripped portion of serum are assayed according to standard RIA methods. The stripped serum serves as a zero reference value to be subtracted from the result obtained with the untouched serum to yield results adjusted for NSI. While this method of stripping a portion of the very serum to be assayed compensates for NSI, it is not wholly satisfactory because it involves an extra cycle of incubation which increases the total time necessary to reach a final assay result after drawing a sample from a patient.
Double antibody PTH RIA's directed at human serum are primarily useful for diagnostic purposes and secondarily useful for research purposes. Similar double antibody PTH RIA's directed at the sera of mammals such as rats, oxen, or cows are particularly useful for advanced research on the functioning of parathyroids, because rats, oxen, and cows can be controlled and manipulated in ways inappropriate for human subjects. It is commonly known that most regions of bovine PTH and hPTH are structurally similar, and that fragments of bovine PTH are present in bovine parathyroid glands and circulate in the blood. Furthermore, it is now known that some regions of rat PTH and hPTH are structurally similar, and that fragments of rat PTH are present in rat parathyroid glands and circulate in rat blood. Because of these structural similarities, some antibodies have a substantially high affinity for bovine PTH, rat PTH, and hPTH. Thus, double antibody PTH RIA's incorporating such antibodies will measure the concentration of bovine PTH and rat PTH as well as hPTH. The effect of NSI on assay results is common to such RIA's whether directed to bovine serum, rat serum, or human serum. It is thought that NSI will similarly effect assays of any mammalian serum. Thus, the various attempts in prior art double antibody PTH RIA's to adjust for the effects of NSI are not wholly satisfactory when such RIA's are directed to mammalian serum.